ABSTRACT
SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.
Subject(s)
Brassinosteroids/metabolism , Indoleacetic Acids/metabolism , Proteome , Signal Transduction , Aminopyridines/metabolism , Arabidopsis , Arabidopsis Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Protein Stability , Proteomics/methods , Succinates/metabolismABSTRACT
Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.
